Bioresorbable compositions of carboxypolysaccharide polyether intermacromolecular complexes and methods for their use in reducing surgical adhesions

ABSTRACT

The present invention relates to improved methods for making and using bioadhesive, bioresorbable, anti-adhesion compositions made of intermacromolecular complexes of carboxyl-containing polysaccharides and polyethers, and to the resulting compositions. The polymers are associated with each other, and are then either dried or are used as fluids. Bioresorbable, bioadhesive, anti-adhesion compositions are useful in surgery to prevent the formation of post-surgical adhesions. The compositions are designed to breakdown in-vivo, and thus be removed from the body. Membranes are inserted during surgery either dry or optionally after conditioning in aqueous solutions. The anti-adhesion, bioadhesive, bioresorptive, antithrombogenic and physical properties of such membranes can be varied as needed by carefully adjusting the pH of the polymer casting solutions, polysaccharide composition, the polyether composition, or by conditioning the membranes prior to surgical use. Bi- or multi-layered membranes can be made and used to provide further control over the physical and biological properties of antiadhesion membranes. Antiadhesion compositions may also be used to deliver drugs to the surgical site and release them locally.

This application is a Division of Ser. No. 08/877,649, filed Jun. 17,1997, now U.S. Pat. No. 5,906,997, issued May 25, 1999.

FIELD OF THE INVENTION

This invention relates generally to the manufacture of membranescomprising carboxypolysaccharide/polyether intermacromolecular complexesand the use of those membranes to prevent adhesions between tissues fromforming after surgery. The membrane properties can be tailored toachieve desired degrees of adhesion prevention, bioresorbability,bioadhesiveness, and antithrombogenic effects.

BACKGROUND OF THE INVENTION

Adhesions are unwanted tissue growths occurring between layers ofadjacent bodily tissue or between tissues and internal organs. Adhesionscommonly form during the healing which follows surgical procedures, andwhen present, adhesions can prevent the normal motions of those tissuesand organs with respect to their neighboring structures.

The medical and scientific communities have studied ways of reducing theformation of post-surgical adhesions by the use of high molecular weightcarboxyl-containing biopolymers. These biopolymers can form hydratedgels which act as physical barriers to separate tissues from each otherduring healing, so that adhesions between normally adjacent structues donot form. After healing has substantially completed, the barrier is nolonger needed, and should be eliminated from the body to permit morenormal function of the affected tissues.

Several different types of biopolymers have been used for this purpose.For example, Balazs et al., U.S. Pat. No. 4,141,973 discloses the use ofa hyaluronic acid (HA) fraction for the prevention of adhesions.However, because HA is relatively soluble and readily degraded in vivo,it has a relatively short half-life in vivo of 1 to 3 days, which limitsits efficacy as an adhesion preventative.

Methyl cellulose and methyl cellulose derivatives are also known toreduce the formation of adhesions and scarring that may developfollowing surgery. (Thomas E. Elkins, et al., Adhesion Prevention bySolutions of Sodium Carboxymethylcellulose in the Rat, Part I, Fertilityand Sterility, Vol. 41, No. 6, June 1984; Thomas E. Elkins, M.D. et al.,Adhesion Prevention by Solutions of Sodium Carboxymethylcellulose in theRat, Part II, Fertility and Sterility, Vol. 41. No. 6, June 1984.However, these solutions are rapidly reabsorbed by the body anddisappear from the surgical site.

In addition to solutions of carboxyl-containing biopolymers, solutionsof polyethers can also decrease the incidence of post-surgicaladhesions. Pennell et al., U.S. Pat. No. 4,993,585 describes the use ofpolyethylene oxide in solutions of up to 15% to decrease formation ofpost-surgical adhesions. Pennell et al., U.S. Pat. No. 5,156,839describes the use of mixtures of carboxymethylcellulose up to about 2.5%by weight, and polyethylene oxide, in concentrations of up to about 0.5%by weight in physiologically acceptable, pH neutral mixtures. Because ofthe neutral pH, these materials do not form association complexes, andthus, being soluble, are cleared from the body within a short period oftime.

The above-described solutions have several disadvantages. First, theyhave short biological residence times and therefore may not remain atthe site of repair for sufficiently long times to have the desiredanti-adhesion effects.

Although the methods of manufacture of certaincarboxypolysaccharide-containing membranes have been described, themembranes are poorly suited for use to prevent adhesions. Butler, U.S.Pat. No. 3,064,313 describes the manufacture of films made of 100%carboxymethylcellulose (CMC) with a degree of substitution of 0.5 andbelow, made insoluble by acidifying the solution to pH of between 3 and5, and then drying the mixture at 70° C. to create a film. These filmswere not designed to be used as anti-adhesion barriers. Anderson, U.S.Pat. No. 3,328,259 describes making films of 100% carboxymethylcelluloseand polyethylene oxide, alkali metal salts, and a plasticizing agent foruse as external bandages. These materials are rapidly soluble in plasmaand water and thus would have a very short residence time as an intactfilm. Therefore, these compositions are not suitable for alleviatingsurgical adhesions.

Smith et al., U.S. Pat. No. 3,387,061 describes insoluble associationcomplexes of carboxymethylcellulose and polyethylene oxide made bylowering the pH to below 3.5 and preferably below 3.0, and then dryingand baking the resulting precipitate (See Example XXXVIII). Thesemembranes were not designed for surgical use to alleviate adhesions.Such membranes are too insoluble, too stiff, and swell to little to beideal for preventing post-surgical adhesions. In addition, theirexcessive acidity would cause tissue inflammation.

Burns et al., U.S. Pat. No. 5,017,229 describes water insoluble filmsmade of hyaluronic acid, carboxymethyl cellulose, and a chemicalcross-linking agent. Because of the covalent cross-linking with acarbodiimide, these films need extensive cleaning procedures to get ridof the excess cross-linking agent; and because they are made without aplasticizer, they are too stiff and brittle to be ideally suited forpreventing adhesions--they do not readily conform to the shapes oftissues and organs of the body.

Thus, there have been few successful antiadhesion membranes. D. Wisemanreviews the state of the art of the field in Polymers for the Preventionof Surgical Adhesions, In: Polymeric Site-specific Pharmacotherapy, A.J. Domb, Ed., Wiley & Sons, (1994). A currently available antiadhesiongel is made of ionically cross-linked hyaluronic acid. Huang et al.,U.S. Pat. No. 5,532,221. Cross-linking is created by the inclusion ofpolyvalent cations, such as ferric, aluminum or chromium salts.Unfortunately, hyaluronic acid (either from natural sources orbioengineered) is quite expensive. Therefore, the prior art discloses nomembranes ideally suited to the variety of surgical uses of the instantinvention. Thus, there are several objects of the instant invention.

A first object is to provide compositions and methods which reduce theincidence of adhesion formation during and after surgery. This includesthe prevention of de novo adhesion formation in primary or secondarysurgery.

An additional object is to prevent reformation of adhesions after asecondary procedure intended to eliminate the de novo adhesions whichhad formed after a primary procedure.

Another object is to provide an inexpensive antiadhesion membrane whichremains intact at the surgical site during the initial stages ofcritical wound healing.

Yet another object of the invention is to provide an antiadhesionmembrane which can hydrate quickly in a controlled fashion to form anintact hydrogel.

An additional object of the invention is to provide an antiadhesionmembrane which is resorbable and completely eliminated from the body.

A further object of the invention is to provide an antiadhesion membranewhich has good handling characteristics during a surgical procedure, isconformable to a tissue, pliable, strong, and easy to mold to tissuesurfaces, and possesses sufficient bioadhesiveness to ensure secureplacement at the surgical site until the likelihood of adhesionformation is minimized.

Yet another objective of the invention is to provide an antiadhesionmembrane with desired properties with drugs incorporated into themembrane, so that the drug can be delivered locally over a period oftime to the surgical site.

To achieve these objectives, the instant invention involves carefullycontrolling the properties of antiadhesion membranes by closelyregulating the pH, amounts of carboxyl residues and polyether within thecarboxypolysaccharide/polyether association complex, to closely controlthe degree of association between the polymers. By carefully controllingthe degree of intermolecular binding and amount of polyether, we canclosely vary the physical properties of the membranes and therefore canoptimize the antiadhesion, bioadhesive, bioresorptive, andantithrombogenic properties of the membranes to achieve the desiredtherapeutic results.

Too much hydration can result in an irreversible transformation of themembrane to a "loose gel" which will not stay in place or willdisintegrate. In addition, too much swelling can create too muchhydrostatic pressure which could adversely affect tissue and organfunction. The membrane must be physiologically acceptable, be soft, havethe desired degree of bioresorbability, have the desired degree ofantithrombogenicity, and must be biologically inert.

SUMMARY OF THE INVENTION

One aspect of the invention is a composition made of anintermacromolecular association of a carboxypolysaccharide (CPS) andoptionally a polyether (PE) which are useful for inhibitingpost-surgical adhesions. Another aspect of the invention comprisesmethods of manufacturing complexes of CPS and PE which exhibit desiredphysical and biological properties.

Creation of complexes with desired properties is accomplished by varyingthe degree of bonding between the polymers. This variation in propertiesis accomplished by varying the pH of the casting solution (hereafterreferred to as "the membrane pH"), the molecular weights of thepolymers, the percentage composition of the polymer mixture, and/or thedegree of substitution (d.s.) by carboxyl residues within the CPS.Additional variation in membrane properties is accomplished byconditioning membranes after their initial manufacture. Multi-layeredmembranes are also an aspect of the invention, with different layersselected to exhibit different properties.

Additionally, in accordance with some aspects of the invention, drugscan be included in the membranes to deliver pharmacological compoundsdirectly to the tissues.

The materials are biocompatible, and are cleared from the body within adesired period of time, which can be controlled. The membranes are usedto inhibit the formation of post-surgical adhesions.

Unlike the prior art, anti-adhesion compositions can be made havingdesired properties. Furthermore, conditioning of anti-adhesion membranesafter their manufacture results in unexpected properties, which areadvantageous for the use of the invention to alleviate post surgicaladhesions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of a theory of formation ofassociation complexes between carboxypolysaccharides and polyethersresulting from hydrogen bonding at different pHs.

FIG. 2 shows the results of studies of pH titrations of the solutionsmade for casting CMC- and polyethylene oxide (PEO)-containing membranes.

FIG. 3 shows the time course of hydration or swelling of CMC/PEOmembranes made from casting solutions at different pHs, from 2.0 to 4.31at room temperature.

FIG. 4 shows the hydration or swelling of CMC/PEO membranes in phosphatebuffered saline (PBS) solution with a pH of 7.4 at room temperature.

FIG. 5 shows solubility in PBS of membranes of different composition andpH.

FIG. 6 shows results of studies of the acidification of PBS solutions byCMC/PEO membranes.

FIG. 7 shows the effect of changing the molecular weight of PEO onhydration or swelling of CMC/PEO membranes.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Definitions

Before describing the invention in detail, the following terms aredefined as used herein.

The term "adhesion" means abnormal attachments between tissues andorgans that form after an inflammatory stimulus such as surgical traum.The terms "adhesion prevention" and "anti-adhesion" means preventing orinhibiting the formation of post-surgical scar and fibrous bands betweentraumatized tissues, and between traumatized and nontraumatized tissues.

The term "association complex" or "intermacromolecular complex" meansthe molecular network formed between polymers containing CPS and/or PE.

The term "bioadhesive" means being capable of adhering to living tissue.

The term "bioresorbable" means being capable of being reabsorbed andeliminated from the body.

The term "biocompatible" means being physiologically acceptable to aliving tissue and organism.

The term "carboxymethylcellulose" ("CMC") means a polymer composed ofrepeating cellobiose units, further composed of two anhydroglucose units(B-glucopyranose residues), joined by 1,4 glucosidic linkages. Thecellobiose units are variably carboxylated.

The term "degree of substitution" ("d.s.") means the average number ofcarboxyl residues present per mole of cellobiose.

The term "discectomy" means a surgical operation whereby a rupturedvertebral disc is removed.

The term "endoscope" means a fiber optic device for close observation oftissues within the body, such as a laparoscope or arthroscope.

The term "fibrous tissue" means a scar or adhesions.

The term "hyaluronic acid" ("HA") means an anionic polysaccharidecomposed of repeat disaccharide units of N-acetylglucosamine andglucuronic acid. HA is a natural component of the extracellular matrixin connective tissue.

The term "hydration" (also "swelling") means the process of taking upsolvent by a polymer solution.

The term "hydration ratio" (also "swelling ratio") means the wet weightof a hydrated membrane less the dry weight divided by the dryweight×100%.

The term "hydrogel" means a three-dimensional network of hydrophilicpolymers in which a large amount of water is present.

The term "laminectomy" means a surgical procedure wherein one or morevertebral lamina are removed.

The term "laparoscope" means a small diameter scope inserted through apuncture wound in the abdomen, used for visualization during minimallyinvasive surgical procedures.

The term "membrane pH" means the pH of the casting solution from whichthe membrane is made.

The term "mesothelium" means the epithelium lining the pleural,pericardial and peritoneal cavities.

The term "peritoneum" means the serous membrane lining the abdominalcavity and surrounding the viscera.

The term "polyethylene oxide" means the non-ionic polyether polymercomposed of ethylene oxide monomers.

The term "tissue ischemia" means deprivation of blood flow to livingtissues.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method of reducing the formationof adhesions during and following surgery comprising the step ofdelivering to a wound an implantable, bioresorbable association complexof carboxypolysaccharides (CPS) and a polyether (PE). Complexes aregenerally made by mixing appropriate amounts and compositions of CPS andPE together in solution, then, optionally acidifying the solution to adesired pH to form an acidified association complex, and then ifdesired, by pouring the solution into a suitable flat surface andpermitting the mixture to dry to form a membrane at either reduced(>0.01 Torr) or normal (about 760 Torr) atmospheric pressure. Theassociation complex is placed between tissues which, during woundhealing, would form adhesions between them. The complex remains at thesite for different periods of time, depending upon its composition,method of manufacture, and upon post-manufacture conditioning. When thetissues have substantially healed, the complex then degrades and/ordissolves and is cleared from the body.

Membranes in accordance with the invention can be made with desireddegrees of stiffness, different rates of bioresorbability, differentdegrees of bioadhesion, different degrees of anti-adhesion effectivenessand different degrees of antithrombogenic properties.

Although the exact mechanism of association complex formation is notcompletely known, one theory is that hydrogen bonding occurs between thecarboxyl residues of the polysaccharide and the ether oxygen atoms ofthe polyether. See Dieckman et al., Industrial and Engineering Chemistry45(10):2287-2290 (1953). FIG. 1 illustrates this theory. The pH of thepolymer solution from which the membrane is cast (the "castingsolution") is carefully titrated to an acidic pH by means of a suitableacid. The initially neutral, anionic polysaccharide carboxyl groups areconverted into protonated, free carboxylic acid groups by the additionof the acid (e.g. hydrochloric acid) to the mixed polymer castingsolution. The protonated carboxyl residues can subsequently bondelectrostatically to the ether oxygen atoms of the polyether, therebyforming hydrogen bonds, a type of dipole-dipole interaction.

Decreasing the pH of the casting solution increases the number ofprotonated carboxyl residues, which increases the number of possiblehydrogen bonds with the polyether. This strengthens the polymer network,and results in a stronger, more durable, less soluble and lessbioresorbable membrane. On the other hand, if the casting solution isnear neutral pH, the carboxyl groups on the carboxypolysaccharide aremore negatively charged and thus repel both each other and the etheroxygen atoms of the PE, resulting in a weakly bonded gel with little orno structural integrity.

For the purpose of illustration, three cases of such interactions can bedistinguished as shown in FIG. 1. The figure shows a schematicrepresentation of the possible intermolecular complexation in which fourcarboxymethyl groups from a carboxypolysaccharide (CPS) chain arealigned opposite to four ether oxygen atoms of a polyether (PE) chain.FIG. 1a shows the situation which would exist at a pH of about 7. Atneutral pH, the carboxyl residues are dissociated, so no hydrogen bondedcomplex is formed between the ether oxygen atoms of the PE and thenegatively charged carboxymethyl groups of CPS. FIG. 1b shows thesituation which would exist at a pH of about 2. At low pH, most of thecarboxyl residues are protonated, so most are hydrogen-bonded to theether oxygen atoms of the PE. FIG. 1c shows the situation which wouldexist at a pH of approximately 3-5. At the pK_(a) of the CPS of about4.4, half of the carboxyl groups are protonated, and thus are hydrogenbonded to the corresponding ether oxygen atoms of the PE. Within thisintermediate pH region, the degree of cross-linking can be carefullyadjusted according to the present invention (FIG. 2).

Membranes made according to FIG. 1b are like those described by Smith etal. (1968). They lack the several key features of the ideal adhesionpreventative membrane. The low pH membranes hydrate poorly. Further,they are rough to the touch, non-pliable, and are poorly soluble.Because they are insoluble, they would not be cleared from the body in asufficiently short time period. Moreover, because of the high acidity ofthe casting solution, they deliver a relatively larger amount of acid tothe tissue compared to more neutral pH membranes. Physiologicalmechanisms may have difficulty in neutralizing this acid load beforetissue damage occurs. Thus, they can have poor biocompatability.

In contrast to the prior art membranes described above, the presentinvention teaches adhesion preventative membranes as schematicallydepicted in FIG. 1c. These membranes are made in an intermediate pHrange, typically between approximately 3 and 5, so that the amount ofcross-linking is neither too great, which would result in complexeswhich would not dissolve rapidly enough, nor too little, which wouldresult in a complex which would disintegrate too rapidly. Furthermore,varying the pH of the casting solutions varies the rheologicalproperties of the solution (Table 1), and varies the physical propertiesof the membranes made from those solutions (Table 2).

The above mechanism for formation of association complexes is notnecessary to the invention. The results of our studies with CPS and PEdescribe the invention fully, without reliance upon any particulartheory of the association between the components.

Manufacturing membranes from CPS/PE casting solutions requires only thatthe solution of CPS and PE can be handled easily. Dilute solutions (upto about 10% weight/volume) of CPS are easy to handle, and solutions ofabout 2% CPS are easier to handle. Solutions of PEO up to about 20%(weight/volume) are possible to make and handle, and solutions of about1% by weight are easy to handle.

The carboxypolysaccharide may be of any biocompatible sort, includingbut not limited to carboxymethyl cellulose (CMC), carboxyethylcellulose, chitin, hyaluronic acid, starch, glycogen, alginate, pectin,carboxymethyl dextran, carboxymethyl chitosan, and glycosaminoglycanssuch as heparin, heparin sulfate, and chondroitin sulfate. Preferably,carboxymethyl cellulose or carboxyethyl cellulose is used. Morepreferably, carboxymethyl cellulose (CMC) is used. The molecular weightof the carboxypolysaccharide can vary from 100 kd to 10,000 kd. CPS inthe range of from 600 kd to 1000 kd work well, and CPS of 700 kd workswell, and is easily obtained commercially.

Similarly, the polyether used is not crucial. The preferred polyether ofthe present invention is polyethylene oxide (PEO). Whereas CMC sodium byitself has been used as an antiadhesion barrier in a gel formulation,CMC/PEO membranes have some unique properties useful for adhesionprevention.

Membranes made of CMC and PEO together are more flexible than membranesmade of CMC alone, which are hard and stiff. The membranes mayaccordingly be manipulated during surgery to conform closely to theshape needed for close adherence to a variety of tissues. Further, theinclusion of PEO in the complex confers antithrombogenic propertieswhich help prevent adhesions by decreasing the adherence of bloodproteins and platelets to the membrane (Amiji, Biomaterials, 16:593-599(1995); Merill, E. W., PEO and Blood Contact in Polyethylene GlycolChemistry-Biotechnical and Biomedical Applications, Harris J. M. (ed),Plenum Press, NY, 1992; Chaikof et al., A.I. Ch.E. Journal36(7):994-1002 (1990)). PEO-containing membranes impair the access offibrin clots to tissue surfaces, even more so than a membrane containingCMC alone. Increasing flexibility of CMC/PEO membranes withoutcompromising the tensile strength or flexibility improves the handlingcharacteristics of the membrane during surgery. The molecular weightrange of the polyether as used in this invention can vary from 5 kd to8000 kd. Polyether in the range from 100 kd to 5000 kd work well and arereadily available commercially.

Varying the ratio of the polysaccharide and polyether altersviscoelastic properties of the solutions (Tables 4, 5), and producesdifferent degrees of adhesion prevention and antithrombogenic effects.Increasing the percentage of CPS increases the bioadhesiveness, butreduces the antithrombogenic effect. On the other hand, increasing thepercentage of PE increases the antithrombogenic effect but decreasesbioadhesiveness. The percentage of carboxypolysaccharide to polyethermay be from 10% to 100% by weight, preferably between 50% and 90%, andmost preferably should be 90% to 95%. Conversely, the percentage ofpolyether may be from 0% to 90%, preferably from 5% to 50%, and mostpreferably should be approximately 5% to 10%.

The tightness of the association and thus the physical properties of theassociation complex between the CPS and PE may be closely regulated.Decreasing the pH of the association complex increases the amount ofhydrogen cross-linking. Similarly, increasing the degree of substitutionof the carboxypolysaccharide in the membrane increases cross-linkingwithin the association complex at any given pH, and thereby decreasesthe solubility and therefore the bioresorbability of the complex.Membranes made from low pH polymer solutions are generally harder andstiffer, dissolve more slowly, and therefore have longer residence timesin tissues than do membranes made from solutions with higher pH or ofhydrogels. Low pH polymer membranes are generally useful in situationswhere the period of adhesion formation may be long, or in tissues whichheal slowly. Such situations may occur in recovery from surgery toligaments and tendons, tissues which characteristically heal slowly.Thus, a long-lasting membrane could minimize the formation of adhesionsbetween those tissues. However, low pH membranes are rough to the touch,crack easily when folded, and tend to shatter easily.

In contrast, membranes made from solutions with higher pH are moreflexible and easier to use than membranes made from solutions with lowerpH. They are more bioadhesive and biodegrade more rapidly than membranesmade at lower pH, and are therefore more useful where the period ofadhesion formation is short. These membranes feel smooth, and arepliable, and are capable of being folded without as much cracking orshattering compared to membranes made from solutions with low pH.

The pH of the association complex of the present invention may bebetween 1 and 7, preferably between 2 and 7, more preferably between 3and 7, even more preferably between 3.5 and 6.0. For certain uses, a pHof about 4.1 is preferred, where there is a desirable balance betweenthe bioadhesiveness, antiadhesion properties, the rates ofbioresorbability and the biocompatability for most of the usescontemplated in the present invention.

Bioadhesiveness is defined as the attachment of macromolecules tobiological tissue. Bioadhesiveness is important in preventing surgicaladhesions because the potential barrier must not slip away from thesurgical site after being placed there. Both CMC and PEO individuallyare bioadhesive (e.g., see Bottenberg et al., J. Pharm.Pharmacol.43:457-464 (1991)). Like other polymers which are known toswell when exposed to water, CMC/PEO membranes are also bioadhesive.

Hydration contributes to bioadhesiveness of membranes (Gurney et al,Biomaterials 5:336-340 (1984); Chen et al., Compositions ProducingAdhesion Through Hydration, In: Adhesion in Biological Systems, R. S.Manly (Ed.) Acad. Press NY (1970), Chapter 10). A possible reason forthis phenomenon is that with increased hydration, more charges on theCMC become exposed, and therefore may be made available to bind totissue proteins. However, excessive hydration is detrimental tobioadhesion. Thus, a means of controlling the bioadhesiveness ofmembranes is to control their hydration properties.

The membranes of the present invention rapidly hydrate in PBS solution(FIG. 3). This behavior mimics that of membranes placed on moist tissuesduring surgery. The hydration of the membranes increases both thethickness of the barrier and its flexibility, thus permitting it toconform to the shape of the tissues to be separated during the periodduring which adhesions could form. The preferred hydration ratios (%increase in mass due to water absorption) for optimum adhesionprevention are 500%-4000%, more preferred ratios are between 700%-3000%,and the most preferred hydration ratio for alleviating adhesions isapproximately 2000% (FIG. 4).

In addition to decreasing the pH of the association complex, increasedintermacromolecular association is achieved using CPSs with increaseddegree of carboxyl substitution. By increasing the density ofprotonatable carboxyl residues on the CPS, there is increasinglikelihood of hydrogen bond formation even at a relatively high pH. Thedegree of substitution must be greater than 0, i.e., there must be somecarboxyl residues available for hydrogen bond formation. However, theupper limit is theoretically 3 for cellulose derivatives, wherein foreach mole of the saccharide, 3 moles of carboxyl residues may exist.Thus, in the broadest application of the invention, the d.s. is greaterthan 0 and up to and including 3. Preferably, the d.s. is between 0.3and 2. CPS with d.s. between 0.5 and 1.7 work well, and CPSs with a d.s.of about 0.65-1.45 work well and are commercially available.

The complexes of the instant invention are intended to have a finiteresidence time in the body. Once placed at a surgical site, the driedmembranes hydrate rapidly, turning into a gel-like sheet and aredesigned to serve as a barrier for a limited time period. Once healinghas substantially taken place, the anti-adhesion barrier naturallydisintegrates, and the components are cleared from the body. The timetaken to clear the body should preferably be no more than 29 daysbecause of increased regulation by the Food and Drug Administration ofdevices intended to remain within the body for more than 30 days.

The mechanisms for bioresorption of CMC/PEO complexes are not wellunderstood. However, an early step in the process of bioresorption issolubilization of the network of CMC and PEO. Thus, increasing thesolubility of the complex increases the ease of clearing the componentsfrom the tissue (FIG. 5). When soluble, CMC and PEO can diffuse into thecirculation and be carried to the liver and kidneys, where they may bemetabolized or otherwise eliminated from the body. Additionally,enzymatic action can degrade carbohydrates. It is possible that enzymescontained in neutrophils and other inflammatory cells may degrade thepolymer networks and thereby increase the rate of elimination of thecomponents from the body.

The degradation and rate of solubilization and disruption of themembrane is manipulated by careful adjustment of the pH during formationof the association complexes, by varying the CPS/PE ratio, and byselecting the appropriate degree of substitution of the CPS andmolecular weights of the PE and CPS. Decreasing the molecular weight ofCPS increases its solubility. (Kulicke et al., Polymer 37(13):2723-2731(1996). The strength of the membrane can be tailored to the surgicalapplication. For example, certain surgical applications (e.g., spine ortendon) may require a stronger, more durable membrane than others (suchas intraperitoneal applications). Manipulation of the above-mentionedexperimental variables allows the manufacture and use of products withvariable residence times in the body.

Biocompatability of the complex of the present invention is a functionof its acidity. A highly acidic complex contributes a relatively largertotal acid load to a tissue than does a more neutral complex.Additionally, the more rapidly hydrogen ions dissociate from a complex,the more rapidly physiological mechanisms must compensate for the acidload by buffering, dilution and other clearance mechanisms. To mimic therate and total amount of acid given up by a membrane in vivo, membranesare placed in PBS solutions and the degree of acidification of the PBSis measured. In addition to membrane pH, membrane composition alsoinfluences the acid load delivered to the body. FIG. 6 and Tables 3 and6 show the results of studies designed to mimic the delivery of acid bymembranes to tissues.

After their manufacture, membranes may be modified to suit theparticular needs of the user. For example, relatively bioresorbablemembranes may be made more insoluble by treating them with solutionscontaining an acid, exemplified, but not limited to hydrochloric,sulfuric, phosphoric, acetic, or nitric acid, the "acidic" method.

Conversely, a relatively non-resorbable acidic membrane may be made morebioresorbable and bioadhesive by conditioning it with alkali such asammonia (the "alkaline" method), or with a buffered solutions such asphosphate buffer (PB) or phosphate buffered saline (PBS; the "buffer"methods). A 10 mM solution of PBS at a pH of 7.4 is preferred, due tothe biocompatability of phosphate buffers. Moreover, the pH of amembrane may be buffered without eliminating the advantages of membranesmade at lower pH. Thus, an originally acid membrane will hydrate slowlyand have a relatively long residence time even if its pH is raised byalkali or buffer treatment.

Table 7 shows the effects of ammonia treatment on properties of CMC/PEOmembranes. A highly acidic original membrane (pH 2.03) acidified a PBSbuffer solution originally at a pH of 7.40 by lowering its pH to 4.33.After soaking this membrane in PBS solution, it hydrated to over 2.5times its original dry weight and after 4 days in PBS, this membranelost approximately 29% of its original mass. In an identical membrane,incubation for 1 min in a 0.5N ammonia solution substantiallyneutralized the membrane so that it released few hydrogen ions into thebuffer solution, and the pH of the PBS solution remained nearly neutral(pH 7.29).

Table 8 shows the effects of phosphate-buffer treatment on properties ofCMC/PEO membranes. Membranes treated with 50 mM phosphate buffersolution for progressively longer time periods had increasingly neutralpH as judged by their decreased release of acid into a PBS solution.Similarly, PBS (10 mM phosphate buffer) neutralized the acid inmembranes (Table 9). Therefore, membranes can be made which arephysiologically compatible with tissues, yet because they are made at anacidic original pH which creates an association complex, the membranesretain the desired properties of the original complex.

Additionally, multi-layered membranes may be made, for example, toincorporate a low pH inner membrane, surrounded by an outer membranemade with a higher pH. This composition permits the introduction of amembrane with long-term stability and low rate of bioresorbability ofthe inner membrane while minimizing adverse effects of low pH membranes,such as tissue damage and the stimulation of inflammatory responses.Moreover, the high pH outer portion is more bioadhesive than low pHmembranes, ensuring that such a membrane remains at the site moresecurely.

Multilayered membranes may also be made which include as one layer, apure CPS or PE membrane. Such a membrane could have the flexibility,antiadhesion, and solubility properties of the side which is a mixtureof CPS and PE, and have the property of the pure material on the other.For example, bioadhesiveness is a property of CPS, and a pure CPS sidewould have the highest degree of bioadhesiveness. Alternatively, a purePE membrane would have the most highly antithrombogenic properties.Thus, a membrane can be made which incorporates the desired propertiesof each component.

Membranes can be made which incorporate drugs to be delivered to thesurgical site. Incorporation of drugs into membranes is described inSchiraldi et al., U.S. Pat. No. 4,713,243. The incorporation may be ateither the manufacturing stage or added later during membraneconditioning prior to insertion. Drugs which may inhibit adhesionformation include antithrombogenic agents such as heparin or tissueplasminogen activator, drugs which are anti-inflammatory, such asaspirin, ibuprofen, ketoprofen, or other, nonsteroidal anti-inflammatorydrugs. Furthermore, hormones, chemotactic factors, analgesics oranesthetics may be added to the membrane, either during manufacture orduring conditioning. Any drug or other agent which is compatible withthe membrane components and membrane manufacture may be used with thepresent invention.

The types of surgery in which the compositions of the instant inventionmay be used is not limited. Examples of surgical procedures includeabdominal, ophthalmic, orthopedic, gastrointestinal, thoracic, cranial,cardiovascular, gynecological, arthroscopic, urological, plastic, ormusculoskeletal.

Between 67% and 93% of all laparotomies and laparoscopies result inadhesion formation. Specific abdominal procedures include surgeries ofthe intestines, appendix, cholecystectomy, hernial repair, lysis ofperitoneal adhesions, kidney, bladder, urethra, and prostate.

Gynecological procedures include surgeries to treat infertility due tobilateral tubal disease with adhesion attached to ovaries, fallopiantubes and fimbriae. Such surgeries including salingostomy, salpingolysisand ovariolysis, Moreover, gynecological surgeries include removal ofendometriosis, preventing de-novo adhesion formation, treatment ofectopic pregnancy, myomectomy of uterus or fundus, and hysterectomy.

Musculoskeletal surgeries include lumbar laminectomry, lumbardiscectomy, flexor tendon surgery, spinal fusion and joint replacementor repair.

Thoracic surgeries which involve sternectomy can be hazardous afterprimary surgery because of adhesion formation between the heart or aortaand sternum. Thoracic surgeries include bypass anastomosis, and heartvalve replacement.

Because many cranial surgical procedures require more than oneprocedure, adhesions involving the skull, dura and cortex can complicatethe secondary procedures.

Ocular surgical uses include strabismus surgery, glaucoma filteringsurgery, and lacrimal drainage system procedures.

General Methods For Testing And Evaluating Antiadhesion Membranes

Hydration Ratio of Membranes

To determine the rate of hydration and the hydration ratio of membranes,pieces of dry membranes, preferably 160 mg, were placed singly in aglass vial and 20 ml phosphate buffered saline solution (PBS, 10 mM, pH7.4, Sigma Chemical Company, St. Louis, Mo.) was added. The membraneshydrate, creating soft sheets of hydrogel. After a certain time period(typically 1 hr to 5 days), each of the hydrated membranes was carefullyremoved from the test vial and placed in a polystyrene petri dish.Excess water was removed using a disposable pipette and by blotting themembrane with tissue paper. Each membrane was then weighed and thehydration ratio (% H) was determined according to the following formula:##EQU1## Solubility of CPS/PE Membranes

To determine the solubility of CPS/PE membranes, we measured therelative solubility in water and the aqueous stability of the membranesas a function of their chemical compositions. Membrane solubility inwater correlates with the resorption time of the membranes in-vivo.

Typically, the test is performed in conjunction with the hydrationmeasurements outlined above. However, the membranes take up salt duringthe hydration test due to exposure to PBS. This added salt results in anartifactually high dry weight. Therefore, after determining thehydration ratio, we soaked the membranes in deionized water (30 ml for30 min.) to remove the salt incorporated in the polymer network. Thewater was decanted and a fresh 30 ml aliquot of deionized water wasadded. The membranes were allowed to soak for another 30 min., weretaken out of the petri dishes, were blotted dry and were placed in agravity convection oven at 50° C. to dry.

The drying time was dependent on the amount of water absorbed by themembrane. Highly hydrated, gel-like membranes took up to 24 hours to drywhereas partially hydrated membranes took as little as a few hours todry. After the membranes lost the excess water, the membranes wereallowed to equilibrate at room temperature for 1-2 hours before weighingthem. The weight measurements were repeated until a constant weight wasobtained. Typically, some rehydration of the membrane took place duringthis period due to adsorption of moisture from the air.

After the desalinization process described above, the membranes wereplaced in petri dishes containing 30 ml deionized water to hydrate forperiods of from 20 minutes to 5 days. Preliminary studies showed thatmembranes at pH within the range of 6 and below did not disintegrateduring the 1 hr desalinization period.

The solubility (S) of membranes was calculated using the followingformula: ##EQU2## The dry mass before soaking is the mass afterdesalinization, and the dry mass after soaking is the mass after thehydration period in water.

Determination of Acid Load Delivered by Membranes

This test was performed in conjunction with the hydration and solubilitytests described above. The test gives an indication of the acid loadwhich the membrane could deliver to a tissue when placed implanted in ananimal or human subject. After manufacture, the membranes were placed ina PBS solution, the complex released protons in a time-dependent wayresulting in a measurable decrease in pH of the PBS solution.

The acid load test was performed using a Model 40 pH meter (BeckmanInstruments, Fullerton, Calif.). 160 mg of dry membrane was placed in aglass vial and 20 ml PBS was added. The initial pH of the PBS solutionwas 7.40; the pH of this solution was gradually decreased as thepolymers in the membrane partly dissolved thereby exposing moreprotonated carboxylic residues. In highly hydrated membranes (pH 4-7)this process was accelerated as the polymer chains were pulled apart bythe hydrostatic forces generated during the hydrating process.

EXAMPLES

In the following examples, carboxypolysaccharide/polyether membranes aredescribed for CMC as an exemplary carboxypolysaccharide, and PEO is theexemplary polyether. It is understood that association complexes ofother carboxypolysaccharides and polyethers can be made and used in thesame ways. Thus, the invention is not limited to these Examples, but canbe practiced in any equivalent fashion without departing from theinvention.

Example 1 Neutral CMC/PEO Membranes

Type 7HF PH (MW approximately 700 kd; lot FP 10 12404)carboxymethylcellulose sodium (CMC) was obtained from the AqualonDivision of Hercules (Wilmington, Del.). PEO with a MW of approximately900 kd was obtained from Union Carbide (Polyox WSR-1105 NF, lot D 061,Danbury Conn.); PEO with a MW of approximately 1000 kd was obtained fromRITA Corporation (PEO-3, lot 0360401, Woodstock, Ill.).

A membrane with a composition of 65% CMC and 35% PEO was made asfollows: 6.5 g of CMC and 3.5 g of PEO was dry blended in a weighingdish. A Model 850 laboratory mixer (Arrow Engineering, Pa.) was used tostir 500 ml of deionized water into a vortex at approximately 750 RPM.The dry blend of CMC and PEO was gradually dispersed to the stirredwater over a time period of 2 min. As the viscosity of the polymersolution increased as the polymers dissolved, the stirring rate wasgradually decreased. After approximately 15 min., the stirring rate wasset at between 60-120 RPM and the stirring was continued forapproximately 5 h to obtain a homogeneous solution containing 2% totalpolymer concentration (wt/wt) without any visible clumps.

Instead of pre-blending the CMC and PEO, an alternative way offormulating the casting solution for the membranes is to individuallydissolve the polymers. The anionic polymer, CMC, can be then acidifiedby adding the appropriate amount of HCl. For example, a 500 ml batch of2% CMC made by dissolving 10.0 g of CMC 7HF in 500 ml deionized waterwas acidified to a pH of 2.6 by adding 2700 μl concentrated HCl("solution A"). Separately, a batch of 2% PEO was made (w/v 900,000 MW,"solution B"). Solutions A and B are then thoroughly mixed in a specificratio using the laboratory stirrer of Example 1 at 60 RPM. The totalpolymer concentration was kept at 2% (w/v), as in Examples 1-2.

Membranes were cast from solutions by pouring 20 g of solution into100×15 mm circular polystyrene petri dishes (Fisher Scientific, SantaClara, Calif. The petri dishes were placed in a laboratory gravityconvection oven set at 40°-45° C., and were allowed to dry overnight at760 Torr. The resulting membranes were carefully removed from thepolystyrene surface by using an Exacto knife.

For larger membranes, 243×243×18 mm polystyrene dishes (FisherScientific) were used. Using the same weight to surface area ratio asfor the circular membranes (in this case, 220 g of casting solution wasused), resulted in a membrane which had a dry weight of approximately4.5 g. The membrane appeared homogeneous, smooth, and pliable. Placing160 mg of this membrane in 20 ml of a PBS solution (pH 7.4) did notchange the pH of the solution. The dry tensile strength and % elongationat break were slightly higher than corresponding membranes which weremade from an acidified casting solution (Table 2). When placed indeionized water or PBS, the membrane exhibited excessive swelling andlost its sheet structure rapidly (within 10 min.) to form a gel-likesubstance which eventually homogeneously dispersed into a polymersolution.

Example 2 Moderately Acidified CMC/PEO Membranes And Hydrogels

The procedure for making acidified membranes in the intermediate pHregion (2.5<pH<7) initially follows the procedure outlined in Example 1.The neutral blended polymer solution containing the polymers specifiedin Example 1 is acidified by adding concentrated hydrochloric acid (HCl,37.9%, Fisher Scientific, Santa Clara, Calif.) while stirring thepolymer solution at 60-120 RPM for 1 hour. Initially, a whiteprecipitate forms in the solution; the precipitate gradually disappearsand a stable solution is formed. Typically, a 2% total polymerconcentration was found useful to achieve the desired viscosity forstable casting solutions. Higher polymer concentrations resulted inpolymer solutions which were too viscous and too difficult to pour.Lower polymer concentrations required more casting solution for the samemembrane weight which greatly increased drying time for equivalentmembranes. In the 500 ml 65% CMC/35% PEO polymer blend of Example 1,1500 μl of concentrated HCl is needed to achieve a pH of 3.1 in thecasting solution. The viscosity of the starting polymer solution droppedby at least 50% by this acidification process.

The titration curves for various polymer blends (as well as 100% CMC and100% PEO) are shown in FIG. 2. FIG. 2 shows the amount of HCl needed tomake casting solutions of desired pHs depending upon the composition ofthe CMC/PEO mixture. Membranes made of 100% CMC (▪) require more acidthan do other compositions to become acidified to the same degree.Increasing the concentration of PEO (decreasing the concentration ofCMC) decreases the amount of acid necessary to acidify a castingsolution to a desired point. Increasing the PEO concentration to 20% hasa small effect, regardless of whether the molecular weight of the PEO is200 k () or 1000 kd (▴). Increasing the PEO concentration to 40% (+) orto 100% (□) further decreases the amount of acid needed to achieve adesired casting solution pH.

Viscosity of Hydrogels

Because the antiadhesion properties of a hydrogel are dependent upon itsviscosity, we determined the relationship between casting solution pHand the viscosity of the hydrogel. We determined the viscosity of PCS/PEsolutions at 22° C. using a Brookfield™ viscometer. Using methodspublished in the brochure Cellulose Gum, Hercules, Inc., Wilmington,Del., (1986), page 28. Briefly, the composition of the solution to betested is selected, and by referring to Table XI on page 29 of CelluloseGum, the spindle number and spindle revolution speed is selected.Viscosity measurements are made within 2 hr after stirring the solution.After placing the spindle in contact with the solution, and permittingthe spindle to rotate for 3 minutes, the viscosity measurement is readdirectly in centipoise on a Brookfield Digital Viscometer (Model DV-II).We studied 65% CMC/35% PEO solutions made with 7HF PH CMC and 1000 kdPEO (RITA) at a pH of 7.5. Another 65% CMC/35% PEO solution was made ata pH of 3.1.

                  TABLE 1                                                         ______________________________________                                        Effect of Casting Solution pH On Hydrogel Viscosity                                         Viscosity @ pH 7.5,                                                                        Viscosity @ pH 3.1,                                  RPM 22° C. (centipoise) 22° C. (centipoise)                   ______________________________________                                        0.5       38,000       13,000                                                   1.0 31,000 12,000                                                             2.0 23,200 10,400                                                             5.0 19,400 8,800                                                              10 15,500 7,300                                                             ______________________________________                                    

Table 1 shows the change in viscosity due to acidificiation of castingsolutions. Reducing the pH from 7.5 to 3.1 decreased the viscosity ofthe casting solution by more than half. Because the viscosity of ahydrogel is related to its ability to prevent adhesions, possibly due toits ability to remain in one site for a longer time period, gels ofhigher pH have greater anti-adhesion properties. Further, it is alsopossible to characterize casting solutions by their viscosity as well astheir pH. Thus, for situations in which the measurement of pH is not beas easy or reliable, measurements of viscosity are preferred. To makemembranes, the acidified casting solutions containing the weaklyH-bonded intermolecular PEO-CMC complex were next poured intopolystyrene dishes and dried out in a similar way as described inExample 1. After drying, physical properties were determined.

Physical Properties of CMC/PEO Membranes

Tensile strength and elongation of membranes are measured for pieces ofmembrane in the shape of a "dog bone," with a narrow point being 12.7 mmin width. The membranes are then mounted in an Instron™ tester equippedwith a one ton load cell. The crosshead speed is set at 5.0 mm/min. Wemeasured membrane thickness, tensile strength, and elasticity (%elongation of the membrane at the break point). Results are reported forthose samples that had failure in the desired test region. Those samplesthat either failed at the radius of the sample or in the grips wereconsidered improper tests and results of those tests were discarded.

                  TABLE 2                                                         ______________________________________                                        Physical Properties of CMC/PEO Membranes                                        Membrane    Thickness  Tensile Strength                                                                        % Elongation                                 Composition (mm) (psi) at Break Point                                       ______________________________________                                        65% CMC/35%                                                                             0.081      6017        4.17                                           PEO (1000 kd), 0.076 5527 4.47                                                pH 3.1 0.076 5956 5.07                                                        65% CMC/35% 0.071 10,568 6.69                                                 PEO (1000 kd), 0.069 10,638 6.61                                              pH 7.5                                                                        80% CMC/20% 0.084 3763 3.20                                                   PEO (5000 kd),                                                                pH 3.1                                                                      ______________________________________                                    

The membranes are all less than 0.1 mm thick. Decreasing the pH of themembrane from neutral decreases the tensile strength, and decreases theelasticity (% elongation) at the break point. Similarly, decreasing thePEO concentration decreases the tensile strength and elasticity of themembranes.

Hydration of CMC/PEO Membranes in PBS

To evaluate the bioadhesive properties of membranes, we determined therate and extent of hydration properties of CMC/PEO membranes accordingto the methods described above.

FIG. 3 shows the time course of hydration of CMC/PEO membranes of thepresent invention. A membrane made of 80% CMC/20% PEO (m.w. 900 kd) at apH of 4.31 rapidly hydrated (). After 2 h in PBS, its hydration ratio(wet wt.-dry wt)/dry wt; % swelling) increased to more than 6000%.After, 5 h in PBS, this membrane's hydration ratio was nearly 8000%.This highly hydrated membrane lost its cohesiveness and substantiallydisintegrated thereafter. Reducing the membrane pH to 3.83 and belowresulted in membranes which hydrated nearly to their equilibrium pointswithin 2 hrs. and maintained their degree of hydration and cohesivenessfor at least 40 hrs. The degree of hydration was dependent upon themembrane pH with the least acidic membranes being capable of swelling toa higher degree. At a pH of 3.83 (▴), the membrane had a hydration ratioof nearly 6000%, whereas at a pH of 2.0 (□), the hydration ratio wasless than 300%. Within the range of pH from 3.2 to 4.3, the degree ofhydration is very sensitive to the pH.

FIG. 4 shows a summary of another study of the effect of membranecomposition and pH on the hydration of CMC/PEO membranes. Hydration wasmeasured after at least 6 hrs in PBS-, a time after which the degree ofhydration had nearly reached equilibrium for each membrane (see FIG. 3).For each of the compositions studied, increasing the membrane pHincreased the hydration of the membrane. Membranes of 100% CMC (▪)increased their hydration ratios from approximately 100% at a membranepH of 1.7 to over 1300% at a membrane pH of 3.4. For membranes made of80% CMC/20% PEO, the molecular weight of the PEO had a slight effect onhydration. Membranes made with 900 kd PEO (▴), hydrated slightly more ata given pH than membranes made with 200 kd PEO (). Furthermore,membranes made with CMC of a higher degree of substition (d.s.=1.2; ⊕)hydrated similarly to those of 100% CMC with a degree of substitution of0.84 (▪). Finally, membranes that were made with 50% CMC/50% PEO (900kd) hydrated less than any of the other membranes, except at lowmembrane pH (<2.5).

Solubility of CMC/PEO Membranes

Because the biodegradation of CPS/PE polymers is related to solubility,we measured the solubility of membranes after at least 4 days in PBSaccording to methods described above. FIG. 5 shows the effects ofmembrane pH and composition on the solubility of membranes in PBSsolution. Membranes were made of different CMC/PEO compositions and atdifferent membrane pHs. For all membranes, as the membrane pH increased,the solubility in PBS increased. Membranes of 100% CMC (▪) were theleast soluble. Membranes containing PEO were more soluble, withmembranes made of 900 kd PEO (▴) being less soluble than membranes of200 kd PEO (). Further increasing the percentage of PEO to 50% (+)further increased membrane solubility. Decreasing the molecular weightof the CMC (7MF;*) increased the solubility. Additionally, increasingthe degree of substitution of the CMC from 0.84 to 1.12 (⊕) resulted ineven more soluble membranes. Also, with the higher degree ofsubstitution, there was a larger effect of pH on membrane solubility.For the other membranes, the effect of increasing pH appeared to be ofsimilar magnitude regardless of the composition of the membrane. Thus,the slopes of the lines were similar. These results indicate thatregardless of membrane composition, the solubility of membranes can beincreased by increasing the membrane pH. Moreover, because bioresorptionrequires solubilization, more highly soluble membranes will be clearedfrom the body more rapidly than less soluble membranes.

Biocompatability Of CMC/PEO Membranes

Because biocompatability is related to the acid load delivered to atissue, we determined the acid load delivered by CMC/PEO membranes to aPBS solution as described above as a suitable in-vitro model. We firstdetermined the time course of acidification of PBS solutions exposed todifferent compositions of CMC/PEO membranes.

                  TABLE 3                                                         ______________________________________                                        Time Course Of Acidification Of PBS By CMC/PEO Membranes                                          Time in PBS Solution (hr)                                         Casting                           45h PBS                               Membrane Solution     pH                                                      Composition pH 1 3.5 21 45 Change                                           ______________________________________                                        80% CMC/                                                                              1.85    6.26    5.62  4.78  4.64  2.76                                  20% PEO 3.17 6.53 5.71 5.61 5.65 1.75                                         (900 kd)                                                                      50% CMC/ 1.77 6.60 6.12 5.62 5.42 1.98                                        50% PEO 2.71 6.47 6.13 6.01 5.98 1.42                                         (900 kd)                                                                      80% CMC/ 1.82 3.71 3.39 3.52 3.45 3.95                                        20% PEO                                                                       (8 kd)                                                                      ______________________________________                                    

Table 3 shows the kinetics of acidification of a PBS solution by CMC/PEOmembranes of the instant invention. When added to a PBS solution,membranes released acid into the solution, thereby lowering the solutionpH. This process occured slowly, with a reduction in solution pH ofapproximately 1 pH unit in the first hour for membranes including thosecombining high molecular weight PEO. This is true for membranes castfrom low pH polymer solutions as well as those cast from higher pHpolymer solutions. The remaining reduction in pH occurred over the next20 hrs, at which time the solution pH remained approximately constant.By 45 hrs in the PBS solution, the pHs have decreased to below 6.0.

Additionally, as the molecular weight of the PEO decreased, the solutionpH decreased more rapidly and to a higher degree than membranes made ofhigh molecular weight PEO. This finding might be due to an ability ofhigher molecular weight PEOs to shield the acidic carboxyl residues ofthe CMC, thereby decreasing the dissociation of carboxyl hydrogen ions.

These results suggest that high molecular weight PEO acts to slow thedelivery of acid to tissues, and thus, protects them from excessiveacidification. Moreover, as protons are released in vivo, they will bediluted in the extracellular spaces, buffered by physiological buffers,and ultimately cleared from the tissue by the lymphatic and circulatorysystems. Over the relatively long time during which protons arereleased, the physiological dilution, buffering, and clearancemechanisms will remove the acid load, keeping the pH at the tissuewithin acceptable ranges. Thus, these membranes are suitable forimplantation in vivo without causing excessive tissue disruption due toa large acid load being delivered.

FIG. 6 shows the results of studies in which the pH of the PBS solutionvaries as a function of the membrane pH and composition of the membrane.Membranes were placed in PBS solution for 4-5 days, times at which theacidification had reached equilibrium (Table 3). The membranecomposition which resulted in the least acidification were thepre-conditioned 80/20/300 k membranes (∘). These membranes were made asdescribed above, except for an additional step of soaking the membranesin PBS and then re-drying them (see Examples 7-9). The 80/20/200 kmembranes cast in PBS (+) delivered the next lowest acid load, and the50/50 CMC/PEO (900 k) series of membranes (Δ) delivered the third lowestacid load to the PBS solution. Membranes made of 100% CMC: (▪),80/20/200 k (), and the 80/20/900 k (▴) delivered progressively moreacid to the PBS, and the 80/20/300 k series of membranes made with CMCwith a degree of substitution of 1.12 delivered the most acid to the PBSsolution.

FIG. 6 also shows that conditioning membranes by soaking them in PBSdecreased the acid load delivered to the PBS solution. For example, apre-conditioned membrane cast at an original pH of 3.4 reduced the pH ofthe PBS solution only to 7.0 from 7.4. Thus, for those applications inwhich a long lasting membrane is needed, but one which will cause theleast acidification, preconditioning of an acidic membrane in PBS isdesirable.

Example 3 Membranes With Different PEO/CMC Ratios

A 500 ml batch of a 80/20 CMC/PEO membrane was obtained by dissolving8.0 g CMC and 2.0 g PEO in 500 mL deionized water (source of CMC andPEO, and solution processes were as in Example 1). While stirring at lowspeed (60 RPM), 200 g of this polymer solution was acidified with 1500μl of 5 N HCl (LabChem, Pittsburgh, Pa.), resulting in an equilibrium pHof 3.17. The acidified polymer solution was next poured into polystyrenedishes and dried out in a similar way as described in Example 1. Bychanging the relative amounts of CMC and PEO, membranes with differentcompositions were obtained. 100% CMC membranes were more brittle andless flexible than PEO-containing membranes. For our purposes, membraneswhich contain more than 70% PEO are generally not preferable as thesemembranes were unstable in an aqueous environment.

                  TABLE 4                                                         ______________________________________                                        Viscosity Of Solutions with Different CMC/PEO Ratios                            (cps, @ Spindle #6, 20° C.)                                            Membrane                                                                      Composition                                                                   (1000 kd PEO)                                                                 (% CMC/% PEO; Spindle RPM                                                   pH)       0.5      1.0     2.5    5.0   10.0                                  ______________________________________                                        25/75                                                                           4.0 8000 7000 4800 4400 3700                                                  2.6 3200 3000 2800 2400 2000                                                  33/66                                                                         4.0 8000 7000 6800 6200 5100                                                2.6       --       3000    3200   2800  2500                                    50/50                                                                       4.0       16,000   15,000  12,800 10,600                                                                              8400                                    2.6 4000 5000 4800 4200 3500                                                  66/33                                                                         4.0 28,000 25,000 20,400 16,000 12,300                                        2.6 8000 7000 6400 5800 4900                                                  100% CMC                                                                      4.0 72,000 61,000 42,800 31,600 28,700                                        2.6 88,000 67,000 42,400 29,400 20,400                                        100% PEO                                                                      (900 kd) 480 300 280 290 290                                                  2.6                                                                         ______________________________________                                    

Table 4 shows the effect of CMC/PEO ratio on solution viscosity.Membranes were made with different percentages of PEO (m.w.: 1,000,000)at two different pHs. Solutions containing higher proportions of CMCwere more viscous than solutions containing less CMC. Furthermore, theless acidic solutions had a higher viscosity than solutions with moreacidity. This relationship held for all solutions except for the 100%CMC solution. At a pH of 2.6, the viscosity was slightly higher than ata pH of 4.0. This was possibly due to the association between CMCmolecules at lower pH.

Larger than expected viscosity decreases were obtained when the twosolutions were mixed. For example, an 85% loss in viscosity was achievedwhen solutions A (pH 2.6) and B were mixed in a 50/50 ratio. At aspindle RPM of 2.5, the starting 2% CMC concentration (w/v), pH 2.6solution had a viscosity of 42,400 cps, the 2% PEO solution had aviscosity of 280 cps. Thus, if viscosity of a mixture is the average ofthe viscosities of the components, we would expect that a 50/50 CMC/PEOsolution would have a viscosity of (42400+280)2=21300 cps (approximatelya 50% viscosity decrease from that of CMC alone). However, the actualCMC/PEO (50/50) solutions had a viscosity of only 4,800 CPS. A similar,more than expected decrease in viscosity was reported by Ohno et al.(Makromol Chem., Rapid Commun. 2, 511-515, 1981) for PEO blended withdextran and inulin.

Further evidence for intermolecular complexation between CMC and PEO isshown by comparing the relative decreases in viscosity caused byacidification for the 100% CMC and CMC/PEO mixtures. Table 4 shows at2.5 rpm, the viscosity of CMC solution remained essentially unchangedwhen the pH was decreased from 4.0 to 2.6. However, for mixtures ofCMC/PEO, the acifidiation caused a large decrease in viscosity. Thedecreases were by 69%, 63%, 53%, and 42% for mixtures of CMC/PEO of66%/33%, 50%/50%, 33%66%, and 25%/75%, respectively.

Thus, there is an intermolecular association between CMC and PEO, which,we theorize, results in PEO molecules becoming interspersed between CMCmolecules, thereby preventing intermolecular bonding between the CMCmolecules. Such a theory could account for the observations, but we donot intend to limit the present invention to any single theory ofmolecular interaction. Other theories may account for the observations.

Next, after manufacturing membranes with different CMC/PEO ratios westudied their hydration, acid load, and solubility properties usingmethods described above.

                  TABLE 5                                                         ______________________________________                                        Effect of CMC/PEO Ratio on Hydration, Acid Load and Solubility                  Membrane                                                                      Composition                                                                   (% CMC 7HF/ Membrane Hydration Acid Load Solubility                           % PEO 900 kd) pH Ratio (%) (PBS pH) (% Mass Loss)                           ______________________________________                                        100% CMC  2.52     1145      3.46   9.7                                         66/33 2.87 2477 3.80 30                                                       50/50 2.94 3077 4.58 34                                                     33/66     2.98     (dissolved)                                                                             5.88   (dissolved)                               ______________________________________                                    

Table 5 shows the effect of increasing the PEO concentration in CMC-PEOmembranes on the % water uptake, acidity, and mass loss. Increasing thePEO content of membranes increases the hydration ratio and solubilityand decreases the acid load delivered to PBS. These results indicatethat as the total amount of CMC in the membrane decreases, the acid loaddecreases.

The effect of a different CMC/PEO ratios is further demonstrated in FIG.5 (solubility vs. membrane pH), and FIG. 6 (membrane acidity vs. PBSsolution pH).

Example 4 Membranes Of Different Molecular Weight PEO

Membranes of PEO's of different molecular weight were made by mixing 2%(w/v) PEO solutions with 2% (w/v) solutions of CMC (type 7HF PH (lot FP10 12404) obtained from the Aqualon Division of Hercules (Wilmington,Del.). PEO's with a molecular weight of 8000 (8K) was obtained asPolyglycol E8000NF from Dow Chemical, Midlands, Mich. The PEO's withmolecular weights of 300,000 (300K), 900,000 (900K), and 5,000,000 (5M)were all from Union Carbide. 2% (w/v) solutions of PEO were made bydissolving 6.0 g of PEO in 300 ml deionized water according to themethods used in Example 1. The CMC stock solution was similarly made bydissolving 10.0 g CMC in 500 ml deionized water. The CMC stock solutionwas acidified by adding 2100 μl concentrated HCl to decrease the pH ofthe casting solution to 3.37.

A 50% CMC/50% PEO (8K) membrane was made by mixing 40.07 g of the CMCstock solution with 40.06 g of the PEO (8K) stock solution. The castingsolution was acidified to a pH of 3.46. A 50% CMC/50% PEO (300K)membrane was made by mixing 39.99 g of the CMC stock solution with 40.31g of the PEO (300K) stock solution and adding sufficient HCl to lowerthe pH to 3.45. A 50% CMC/50% PEO(900K) membrane was made by mixing39.22 g of the CMC stock solution with 39.63 g of the PEO (900K) stocksolution and adding sufficient HCl to lower the pH to 3.56. A 50%CMC/50% PEO (5M) membrane was made by mixing 38.61 g of the CMC stocksolution with 40.00 g of the PEO (5M) stock solution and addingsufficient HCl to lower the pH to 3.55.

Membranes made from these various acidified CMC/PEO mixtures were castand dried according to the methods given in Example 1. FIG. 7 shows theeffect of the molecular weight of PEO on the hydration ratios of theresulting membranes. The results indicate that increasing the molecularweight of PEO increases the hydration ratio, although there was littleincrease in hydration by increasing the PEO molecular weight from 900 kdto 5000 kd. Further differences between the membranes made from variousmolecular weights of PEO's can be observed from the data presented inFIGS. 4-6.

Example 5 Membranes Of Different Molecular Weight CMC

A 50% CMC/50% PEO membrane was made from CMC (type 7MF PH; lot FP1012939, obtained from the Aqualon Division of Hercules, Wilmington, Del.)and PEO with a molecular weight of 900,000 (Union Carbide). In contrastto the "high viscosity", type 7HF CMC, the 7 MF CMC has a much lowerviscosity in solution. The average molecular weight of type 7 MF isapproximately 250 kd as compared to 700 kd for the 7HF type CMC. 5.0 gof CMC and 5.0 g of PEO (900K) were pre-blended dry and then dissolvedin 500 ml deionized water according to the methods of Example 1. Thesolution was acidified with 950 μL of concentrated HCl which reduced thepH to 3.48. A membrane made from 20.0 g stock casting solution. Otherportions of the stock solution were used to make more acidic membranes(with casting solutions pH's of 3.07, 2.51, and 1.96). The membraneswere cast and dried from these acidified solutions. After drying, thehydration ratio, mass loss, and acid load were determined as previouslydescribed. See Table 6.

                  TABLE 6                                                         ______________________________________                                        Properties of Low Molecular Weight CMC                                          Membrane pH                                                                   50% CMC (7MF)/ Mass Loss Hydration Ratio                                      50% PEO (900 kd) (%) (%) PBS Solution pH                                    ______________________________________                                        3.48        dissolved                                                                              not determined                                                                            5.93                                           3.07 dissolved not determined 5.33                                            2.51 dissolved not determined 5.20                                            1.96 60 343 4.33                                                            ______________________________________                                    

When placed in PBS solution for 5 days (the "acid load" test, seeabove), each of the membranes lowered the pH of the PBS solutions. The 3higher pH membranes lost there sheet-like structure and turned into anamorphous, diffuse gel. Only the most acidic membrane maintained itsstructural integrity. Comparing this membrane with others (FIG. 5) showsthat at a pH of 2.0, the membrane made of lower molecular weight CMC wasthe most soluble. Thus, the strength of the association complex isdependent upon the molecular weight of the CMC.

Example 6 CMC/PEO Membranes With A Different Degree Of CMC Substitution

CMC/PEO membranes were made from CMC of type 99-12M31XP (lot FP10 12159,degree of substitution (d.s.) of 1.17, obtained from the AqualonDivision of Hercules, Wilmington, Del.) and from PEO with a molecularweight of 300,000 (Union Carbide). 200 ml of blended polymer solutionwas acidified with 600 μl of concentrated HCl to yield a stock solutionwith a pH of 4.07. 20.7 g of this casting solution was poured into apetri dish; the membrane was dried as described in Example 1. The restof the stock solution was used to make membranes with increased acidity.The pHs of the casting solutions for those membranes were 3.31, 3.03,2.73, 2.44, and 2.17, respectively.

FIGS. 4-6 show the properties of these membranes compared to others withdifferent compositions of CMC and PEO. FIG. 4 shows that the hydrationratio of CMC with a degree of substition of 1.12 (⊕) is similar to thatof other CMC/PEO membranes with a hydration ratio of 836% water whenplaced in PBS for 4 days. However, there are differences in othermeasured properties. FIG. 5 shows that compared to the other membranes,the membranes made from CMC with the higher degree of substitutionproduce the most soluble membranes. FIG. 6 shows that membranes madefrom highly substituted CMC produce membranes which deliver the largestacid load to PBS. This is consistent with the idea that at any given pH,there are more hydrogen ions available for dissociation in thesemembranes made with higher d.s.

Example 7 Ammonia Conditioning Of Membranes

To study the effects of alkali conditioning on CMC/PEO membranes, 3pieces of dried membranes (approximately 160 mg, composition: 80% CMC(7HF PH)/20% PEO (300K or 5000 kd) were placed in a petri dish. 30 ml of0.5 N ammonium hydroxide (made from 10×dilution of 5 N ammonia, LabChem,Pittsburgh, Pa.) was added, immersing the membranes. Once completelyimmersed, the membranes were allowed to soak for either 1 or 5 min. Themembranes were then removed from the ammonia solution, the excessammonia was blotted off with filter paper, and the membranes were placedin a gravity convection oven at 45° C. and allowed to dry. After dryingand re-equilibrating at room temperature, the membrane's mass wasdetermined. After drying, the membranes hydration ratio, acid load, andsolubility were determined. Results are shown in Table 7.

                  TABLE 7                                                         ______________________________________                                        Effect of Ammonia Conditioning On CMC/PEO Membranes                                                             Mass Mass                                     Membrane    Loss Loss Total                                                   Composition Treatment Hydration PBS after after PBS Mass                      80% CMC/ Control or Ratio pH; NH.sub.3 (4d) Loss                              20% PEO 0.5 N NH.sub.3 (%) at 4 d (%) (%) (%)                               ______________________________________                                        300 kd PEO                                                                             Control  258      4.33 --   29     29                                  pH 2.03 1 min 374 7.29 22 1 23                                                 5 min 368 7.29 22 0 22                                                       300 kd PEO Control 281 3.92 -- 26 26                                          pH 2.45 1 min 551 7.23 21 7 28                                                5000 kd PEO, Control 553 4.24 -- 36 36                                        pH 3.1 1 min 4774 6.98 21 61 63                                             ______________________________________                                    

Table 7 shows that ammonia treatment substantially decreased the acidload delivered to a PBS solution. By extension, this effect would alsodecrease the acid load delivered to a tissue in vivo. Also, compared toother membranes delivering the same acid load to the PBS othersolutions, ammonia-conditioned membranes have lower solubility, andthus, increased residence time in vivo. Therefore, it is possible tointroduce antiadhesion membranes with long residence times which deliverlittle residual acid to tissues. In contrast, unconditioned membranes ata pH of approximately 7.0 rapidly disintegrate, and thus are of littlevalue in preventing post surgical adhesions.

Treating the membrane after initial manufacture reduced the acid load ofthe membrane. Compared to the controls (not soaked in ammonia) in allcases the conditioning treatment increased the pH from approximately 4to more neutral pH values. Compared to the controls, the conditioningtreatment also increased the hydration ratio of the membranes. Whereasthis hydration increase was relatively small for the two types of acidicmembranes, the least acidic (pH 3.1 80% CMC/20% PEO (5M)) membraneswelled to a higher degree. The effect of the treatment therefore isdependent on the prior condition of the membrane. The total mass lossdue to the ammonia conditioning in two cases (for the 80% CMC/20% PEO(300 kd) pH 2.03 membranes) is slightly lower than that of the controls.This unexpected result may be due to the initial loss of salt in theammonia solution followed by a uptake of salt in the salt-depletedmembranes during soaking in PBS.

Example 8 Conditioning Membranes Using Phosphate Buffer

Similar to Example 7, membranes were conditioned after manufacture inphosphate buffer (50 mM, pH 7.40). A piece of dry membrane (0.163 g; 80%CMC (7 HF PH)/20% PEO (5000 kd), pH 3.1) was placed in a petri dish. Themembrane was soaked for 5 min in 30 ml of monobasic potassiumphosphate/sodium hydroxide buffer (50 mM, pH 7.40; Fisher Scientific).After 5 minutes the membrane was removed from the solution, excessbuffer blotted off with filter paper, and the membrane was placed in agravity convection oven at 45° C. to dry. After drying andre-equilibration at room temperature, the membrane's mass was 1.42 g(i.e., 13% mass loss). Other membranes were soaked for 20 or 60 minutesin buffer before drying. After drying, the membranes were tested asabove. The hydration ratio, acid load, and solubility (after 4 days inPBS) for each of those membranes was determined, and the results areshown in Table 8.

                  TABLE 8                                                         ______________________________________                                        Effect Of Phosphate Buffer Conditioning On CMC/PEO Membranes                                                    Mass Mass                                     Membrane    Loss Loss After Total                                             Composition  Hydration PBS After PBS Mass                                     80% CMC/  Ratio ph PO.sub.4 (3 d) Loss                                        20% PEO Treatment (%) (3 d) (%) (%) (%)                                     ______________________________________                                        PEO      Control  258      4.33 --   29     29                                  (300 kd) 5 min 296 5.92 20 10 30                                              pH 2.03                                                                       PEO Control 553 4.24 -- 36 36                                                 (5000 kd) 5 min 572 6.58 13 18 31                                             pH 3.1 20 min 685 7.17 16 19 35                                                60 min 833 7.30 20 17 37                                                   ______________________________________                                    

Table 8 shows that like ammonia conditioning, phosphate bufferconditioning neutralized the acid load delivered to the PBS solution.Moreover, increasing the duration of exposure to phosphate bufferresulted in progressive neutralization of the acid in the membranes. ThepH increased from approximately 4.3 to 7.30 after 1 hour incubation.These membranes remain intact in PBS for at least 3 days. In contrast,membranes made at an original pH of 7.0 and above hydrated rapidly asand completely dissociated and lost integrity within several hours.Thus, conditioning acidic membranes with alkali or neutral phosphatebuffer can decrease membrane solubility (increase residence time invivo) while maintaining a highly biocompatible pH. Further, it isanticipated that soaking acidic membranes in other neutral or alkalinebuffer solutions (e.g., a pH 9.0 boric acid-KCl, NaOH, 0.1 M; FischerScientific) will also be effective in reducing the acidity of anoriginally membrane.

Example 9 Conditioning Membranes Using PBS

To determine whether an isotonic, phosphate buffered saline solution canreduce the acid load delivered by a membrane, we repeated the aboveexperiment as in Example 8, but using PBS as the buffer(10 mM, pH 7.4, 3washes, 20 min each). A piece of dry membrane (wt, 0.340 g; composition:80% CMC (7HF PH)/20% PEO (300 kd); pH of 3.1) was placed in a petri dishcontaining 50 ml of a phosphate buffered saline (PBS) solution (10 mM,pH 7.40, Sigma Chemical Company, St. Louis, Mo.) and allowed soak for 20min. The soaking procedure was repeated another 2 times by decanting thesolution from the membrane and adding fresh PBS. Next, the membrane wasremoved from the PBS solution, blotted and dried as above. After dryingand re-equilibrating at room temperature, the membrane's mass was 0.274g. (a 19.4% mass loss). After drying, the hydration ratio, acid load,and solubility were determined as above. Results are shown in Table 9.

                  TABLE 9                                                         ______________________________________                                        Effect of Phosphate Buffered Saline Conditioning on                             CMC/PEO Membranes                                                                                                    Mass                                   Membrane     Loss                                                             pH    Mass Loss After Total                                                   80% CMC/  Hydration PBS After PBS PBS Mass                                    20% PEO  Ratio pH Conditioning (3 d) Loss                                     (300 kd) Treatment (%) (3 d) (%) (%) (%)                                    ______________________________________                                        3.72    PBS      3230     7.0  20      53   73                                  3.14 PBS 1295 6.02 19 37 56                                                   2.85 Control 362 4.28 -- 32 32                                                2.35 PBS 417 5.26 24 9 33                                                     1.84 PBS 267 5.14 23 2 25                                                   ______________________________________                                    

As with phosphate buffer, conditioning acidic membranes with PBS raisesthe membrane pH without completely disrupting the strong associationbetween polymers that originally existed at the lower pH. Thus, anoriginal membrane of pH 3.14, when conditioned using the PBS buffermethod and subsequently placed in PBS, generated a pH of 6.02. Anon-conditioned membrane which generates the same pH in PBS wouldoriginally have a pH in the range of 3-4. Additionally, except for pHsbelow 2, the conditioned membranes hydrate to a higher degree thanun-conditioned membranes. Thus, the conditioned membranes retain someproperties of the original, acidic membranes, yet are more biocompatibledue to the decreased acid load delivered in solution.

Example 10 Multilayered CMC/PEO Membranes

To provide membranes with more varied properties, membranes were made bysandwiching an acidified membrane between two layers of a neutralmembrane, the latter of which may or may not have the same CMC/PEO ratioas the acidified membrane. A sheet of partially dried neutral membranewas first placed on a dry flat surface used as the drying surface forthe laminated membrane. A sheet of partially dried acidified membrane ofslightly smaller dimensions was carefully placed on the neutralmembrane. Next, another sheet of partially dried membrane was carefullyplaced over the acidified membrane such that the edges of the twoneutral membranes were aligned and that none of the acidified membraneextended beyond the edges of the two neutral membranes. When all thethree sheets were properly aligned, deionized water was slowlyintroduced into the petri dish, with care being taken not to misalignthe sheets relative to one another. When all sheets were wetted, anon-absorbable porous thin membrane such as a nylon filter medium wascarefully placed over the wetted laminate and only slightly pressed ontoit. This assembly was then left undisturbed until it is dry, at whichpoint the porous membrane was carefully removed followed by removal ofthe laminated membrane from the flat surface.

An alternative, double-layered membrane was made in a similar fashion.The bi-layered membrane exhibits different properties on each side. Thelow pH side, which is more poorly bioadhesive, permits that side toslide more easily over a tissue than the side with higher pH. The sidewith higher pH would adhere more strongly to the tissue in contact withit and conform to the crevices in the tissue better keeping it in place.Such membranes are valuable in situations where a mobile tissue normallycan move freely with respect to a more fixed tissue.

Another bi-layered membrane was made by placing a partially driedmembrane (ratio of CMC:PEO=95:5, pH 3.0, cast from 15 gm of a 2% polymersolution) in a petri dish and then pouring a CMC/PEO (ratio ofCMC:PEO=95:5, pH 5.5, cast from 10 gm of a 2% polymer solution) mixtureon top of the partially dried membrane. The mixture and partially driedmembrane were then dried together to form the final, bi-layeredmembrane. In a similar way, bilayered membranes of varying PEOcompositions were made, e.g., membranes in which the two layers havedifferent PEO contents. The higher the PEO content of the layer, themore slippery the surface of that layer becomes. The other layer, withlower PEO content, adheres more strongly to the tissue.

An example is abdominal surgery, where the intestinal membranes movefreely with respect to each other and to the surrounding abdominalperitoneum. Additional examples involve thoracic surgery, where thelungs must be able to move with respect to the surrounding peritoneum.Placing the high pH side of a membrane against the parietal peritoneumwill keep it in place but will permit the visceral peritoneum attachedto the lungs to move freely. Similarly, in cardiac surgery, placing thehigh pH side of a bilayered membrane onto the pericardium will keep themembrane in place and permit the low pH side to slide more freely overcardiac tissues, for example, the myocardium. Similarly, in orthopedicsurgery, placing the high pH side of a membrane against a fixed tissue,such as bone or periosteum, will cause it to adhere more firmly to thoselocations and permit a less fixed tissue, such as a ligament, tendon, ormuscle, to move more freely.

Example 11 Effect of Concentration of CMC/PEO On Stability Of CastingSolutions

To determine the effects of the CMC and PEO concentrations on thestability of casting solutions, we added 16 g of CMC d.s.=1.2. and 4 gPEO (300 kd) to 50 ml isopropanol to make a slurry, which was then addedto 450 ml water. This resulted in a relatively homogeneous but moreviscous casting solution than that of Examples 1-10. A series ofmembranes were made by acidifying portions of the casting solution toprogressively lower pHs. 11 g portions of the casting solution werepoured into 10 cm petri dishes and dried.

Membranes were homogeneous above pH of about 3.3, whereas theassociation complexes precipitated from the casting solution at lowerpH. At lower membrane pH, the resulting membranes had areas ofinhomogeniety and holes, and had rough surfaces.

Membranes can be made from solutions of CMC as high as 10% by weight andof PEO as high as 20% by weight.

Example 12 Antithrombogenic effect of CMC/PEO Membranes

Samples of CMC (7 HF PH) and CMC/PEO (5000 kd) membranes were made withCMC/PEO ratios of 80%/20%, 65%/35%, and 50%/50%. An observation chamberfor adherent platelets was assembled consisting of a polymer-coatedglass slide, two polyethylene spacers, and a glass coverslip. Humanblood, obtained from healthy adult volunteers after informed consent,was collected in heparin-containing evacuated containers (Vacutainers™,Becton-Dickinson, Rutherford, N.J.). Heparinized blood was centrifugedat 100 g for 10 min to obtain platelet-rich plasma (PRP).

Two hundred μl of PRP was instilled into the platelet observationchamber. Platelets in PRP were allowed to adhere and activate on thepolymer surfaces for 1 hr at room temperature. Non-adherent plateletsand plasma proteins were removed by washing the chamber with PBS.Adherent platelets were fixed with 2.0% (w/v) glutaraldehyde solution inPBS for 1 hour. After washing with PBS, the platelets were stained with0.1% (w/v) Commassie Brilliant Blue (Bio-Rad, Hercules, Calif.) dyesolution for 1.5 hours. Stained platelets were observed using a NikonLabophot™ II light microscope at 40×magnification (Melville N.Y.). Theimage of adherent platelets was transferred to a Sony Trinitron™ videodisplay using a Mamamatsu CCD™ camera (Hamamatsu-City, Japan). TheHamamatsu Argus-10™ image processor was used to calculate the number ofplatelets per 25,000 μm² surface area in every field of observation. Theextent of platelet activation was determined qualitatively from thespreading behavior of adherent platelets. Images of activated plateletswere obtrained from the Sony Trinitron™ video display screen using aPolaroid ScreenShooter™ camera (Cambridge, Mass.).

The number of adherent platelets and the extent of platelet activationare considered early indicators of the thrombogenicity ofblood-contacting biomaterials. Platelet activation was measuredqualitatively by the extent of platelet spreading on the polymersurfaces. The extent of platelet spreading was judged from 1 (leastreactive) to 5 (most reactive) as described in Table 10.

                  TABLE 10                                                        ______________________________________                                        Evaluation of Platelet Activation: Surface-Induced Spreading                    Platelet Approximate                                                          Activation Spread Area                                                        Stage (μm.sup.2) Remarks                                                 ______________________________________                                        1      10-15       Contact-adherence. Platelets not active.                     2 15-25 Partially active. Initiation of pseudopods.                           3 25-35 Partially activated. Pseudopod extension                                and initiation of release of granular                                         contents.                                                                   4 35-45 Partially activated. Significant pseudopod                              formation and extension. Complete                                             release of granular contents.                                               5 >45 Fully activated. Retraction of                                            pseudopods leading to the flat or                                             "pancake" shape.                                                          ______________________________________                                    

                  TABLE 11                                                        ______________________________________                                        Platelet Adherence And Activation By CMC/PEO Membranes                                          Number of Adherent                                                                            Extent of                                     Membrane Composition Platelets (per 25,000 μm.sup.2).sup.a Activation    ______________________________________                                        100% CMC      95.8 ± 15.3  2.96 ± 0.37                                    80% CMC/20% PEO 48.1 ± 10.9 3.25 ± 0.35                                 65% CMC/35% PEO 17.8 ± 4.25 1.57 ± 0.39                                 50% CmC/50% PEO 5.25 ± 2.67 1.00 ± 0.00                               a: mean ± standard deviation (n = 24).                                     ______________________________________                                    

Table 11 shows that significant number of platelets had adhered andactivated on membranes made of 100% CMC. On the average, more than 95activated platelets were present per 25,000 μm². The number of adherentplatelets and the extent of activation decreased with increasing PEOcontent in the membranes. The CMC/PEO 50%/50% membranes had the leastnumber of platelets. On the average, only 5.0 contact-adherent plateletswere present on these membranes.

The results of this study indicate that CMC/PEO membranes, especiallythe 50%/50% CMC/PEO membrane, is highly anti-thrombogenic, based on thereduction in the number of adherent platelets and the extent of plateletactivation on these surfaces. Thus, increasing the amount of PEO inmembranes increases their antithrombogenic properties.

To determine whether CMC and PEO adversely affect blood clotting invivo, we performed a series of studies in which we injected rabbits withCMC/PEO mixtures, and measured prothrombin time.

Four rabbits (2.4 to 2.8 kg) were anesthetized using ketamine (40 mg/kg)and xylazine (8 mg/kg), and 0.20 ml of clinical grade 2% CMC, 0.05% PEO,50% H₂ O and 47.9% balanced salt solution (Lot #SD011089) was injectedinto the lower spinal area using a 27-gauge, 1/2 inch needle. A fifth,uninjected rabbit (2.8 kg) served as the control. Blood samples(approximately 1.6 ml) were taken at 0 (before injection), 2, 6, 24, 48,and 96 hr postdose. To 1.6 ml of the collected blood, 0.2 ml of 3.8%sodium citrate solution was added. After mixing plasma was prepared bycentrifuging the sample at 2000 rpm for 3 to 5 minutes in a clinicalcentrifuge. Plasma was pipetted into a separate labeled tube and kept onice. The sample was frozen and sent to California VeterinaryDiagnostics, Inc., West Sacramento, Calif. for prothrombin-timedetermination, which was conducted in compliance with FDA's GoodLaboratory Practice Regulations.

Table 12 shows the prothrombin times for each sample of rabbit plasma atvarious sampling times. Rabbit blood coagulates more quickly than humanblood (Didisheim et al., J. Lab. Clin. Med. 53, 866-1959); thus, severalof the samples collected from these rabbits coagulated before analysis.However, the samples assayed showed no effect of the CMC/PEO mixture onthe prothrombin time except for rabbit No. 3, which showed a transientincrease but recovered by day 4.

                  TABLE 12                                                        ______________________________________                                        Prothrombin Time (Seconds) of Rabbits                                           Injected with CMC/PEO                                                                     Rabbit Number                                                   Time (hr) 1         2     3        4   5*                                     ______________________________________                                        0         7.2       7.2   7.1      8.4 7.1                                      2 --  7.1 7.1 7.1 7.1                                                         6 7.3 7.1 7.1 7.8 7.1                                                         24 7.2 7.1 10.6 7.1 8.0                                                       48 7.3 -- 10.3 -- --                                                          96 6.2 6.5 6.5 6.0 6.0                                                      ______________________________________                                         *Control rabbit not injected with CMC/PEO.                                    -- indicates that assay was not performed because the sample had              coagulated.                                                              

Example 13 Determination of Bioadhesiveness of CMC/PEO Membranes

Bioadhesiveness of membranes was determined generally using a peel testdescribed below. Several membranes composed of CMC(7HF PH) and PEO(molecular weight 5000 kd) and varying in acidity were tested for theirrelative bioadhesiveness using an in vitro test. Fresh, center-cut porkchops purchased from a local store were used as adherends to themembranes. Six thinly cut pork chops were placed in polystyrene bioassaydish (243×243×18 mm) and some water placed in the dish to keep arelatively moist environment. Care was taken to blot off any excesswater from the exposed side of the pork chop. Six membranes were cut ina rectangular shape to a mass of 120-130 mg and subsequently placed onsix individual pieces of meat with their smooth sides down. The smoothside of the membrane is that side which was attached to the polystyrenesurface during the drying process. The other side of the membrane whichwas exposed to air generally yields a slightly rougher surface. A topcover of polystyrene was placed over the dish and the membranes wereallowed to hydrate and adhere to the meat at room temperature for 3hours. In a similar manner, other bioassay dishes were used to testother membranes.

After the 3 hour incubation period, the membranes and the meat werecarefully examined in a qualitative way for clarity (color,transparency), structural character of the membrane, form of themembrane (folding on the meat), blanching, rippling as a result ofstrong bioadhesion. The adhesion force in gm. was measuredquantitatively in a peel test by first attaching a clip to the edge ofthe membrane, subsequently attaching the clip to a spring scale (0-10 gmor 0-250 gm range) and slowly pulling the membrane off the meat byvertically raising the spring scale. The force in gm. needed to pull themembrane completely free of the meat, or in some cases, to cause a ripin the membrane was recorded.

                  TABLE 13                                                        ______________________________________                                        Summary: Comparative Adhesion Strength of                                       CMC/PEO Membranes                                                                        % PEO (5000 kd) in Membrane                                      Membrane pH                                                                            35%      20%     10%   5%    2.5%  0                                 ______________________________________                                        2.00     --       2       --    --    --    100                                 2.80 7 7.5a -- -- -- 0                                                        3.00 9 7.5.sup.a 7.sup.b 120.sup.b 50.sup.b 9                                 3.10 -- 83.sup.b 6.sup.b --  --                                               3.30 -- -- -- >150.sup.b 67.sup.b 11.sup.b                                    4.00 -- -- 8.sup.c 10.sup.c 7.sup.c 3                                       ______________________________________                                         .sup.a : mean value: n = 2 ea                                                 .sup.b : mean value: n = 3 ea                                                 .sup.c : mean value: n = 4 ea                                            

The results shown in Table 13 show that the adhesion force betweenCMC/PEO membranes is related to the membrane pH. The pH showing thegreatest adhesive force for a given PEO percentage was approximately3.30, but either increasing or decreasing the pH from this leveldecreased adhesion force. Further, the adhesion force was related to the% PEO in the membrane. The membranes with the highest PEO percentageexhibited the least adhesion. Increasing the PEO percentage increasedadhesion until 5% PEO is reached, but further increases in PEOconcentration decreased adhesive force.

Example 14 In Vivo Clearance of CMC and PEO

To determine the in vivo clearance of CMC and PEO, we performed a seriesof experiments in which we injected rats with radio-labeled CMC and PEO(2% CMC, 0.05% PEO, 50% H₂ O and 47.9% balanced salt solution). Thestudies were conducted under Good Laboratory Practices.

Formulations containing [¹⁴ C]carboxymethylcellulose (CMC) and [¹⁴C]polyethylene oxide (PEO) were injected into the lower spinal area offour groups of six rats (3 male, 3 female); two groups were sacrificedafter 3 days and the remaining two groups after 7 days. Urine and feceswere collected daily from these rats to study the excretion pattern ofthe radioactivity. In addition, representative internal organs wereassayed for the residual levels of radioactivity in these rats. Twoseparate sets of six rats were similarly injected, and blood sampleswere assayed for radioactivity at 0-time (pre-injection) and 8, 24, 48,72, 96, and 168 hours after injection.

Both compounds were excreted primiarily in the urine. Most of theexcretion in urine occurred during the first 24 hours. In the 7-daystudy, the half-times for excretion of the ¹⁴ C-CMC in the urine andfeces were approximately 0.2 day (5 h) initially followed by a longerexcretion half-time of approximately 1.6 days. The corresponding valuesfor ¹⁴ C-PEO were 0.2 day (5 h) and 1.7 days, respectively. Of theorgans assayed, the liver and kidney contained the highest levels ofradioactivity. The percentage of the injected dose in the liver wascomparable for ¹⁴ C-CMC and ¹⁴ C-PEO but that in the kidney was at least6 times higher after injection of ¹⁴ C-PEO than after injection of ¹⁴C-CMC.

The radioactivity level in the blood after ¹⁴ C-CMC administrationdeclined with half-time of approximately 1 day, whereas the bloodhalf-time for ¹⁴ C-PEO was approximately 4 days. Higher percentages ofthe administered dose remained in the carcass plus injection site for ¹⁴C-CMC than for ¹⁴ C-PEO. The mean overall recovery of the administereddose was 80+% for both compounds. No adverse reactions to the injected¹⁴ C-CMC or ¹⁴ C-PEO were observed.

Example 15 Bioresorbability of CMC/PEO Membranes

The bioresorbability of CMC/PEO membranes is determined by making asurgical incisions in the rear legs of rats, and placing a portion of aCMC/PEO membrane into a muscular layer. Several membranes of differentcomposition or degree of cross linking are inserted into each animal,after which the incisions are closed. A sufficient number of animals areto be used for each type of membrane to be evaluated. Daily thereafter,animals are sacrificed, the incisions re-opened and the remainingmembranes are observed for the degree of intactness, and theirlocations. Membranes are removed, blotted to remover excess water,weighed while wet, re-dried, and re-weighed. The amounts of fluidabsorbed, of solids remaining, and the appearance of the membranes arenoted. Comparisons are made between the length of time in situ, tissuelocation, the membrane composition, pre-insertion conditioning, and theresorbability are made. The membranes of the instant invention aretailored to have a desired degree of bioresorbability.

Example 16 Determination of Antiadhesion Properties of CMC/PEO Membranes

The ability of CMC/PEO membranes to inhibit adhesion formation isdetermined according to the standard method of Harris et al., Surgery117(6):663-669, (1995). Adult rats are used. They are anesthetized withintraperitoneal sodium pentobarbital (43 mg/kg) until deep surgicalanesthesia was achieved, as determined by absence of pain responses topaw pinching and the absence of eyelid reflexes. They are placed ventralside up, and their abdominal hair is removed, and the skin is cleanedusing iodophor scrub and rinsed with 70% alcohol.

Under sterile conditions, a 6 cm long ventral midline incision is madeand the skin retracted. A 4 cm long midline incision is made in theabdominal wall, and the right abdominal was is reflected. A 1 by 2 cmsegment of the parietal peritoneum is excised, including a superficiallayer of underlying muscle, 1 cm lateral to the midline incision. Thecaecum is then elevated so that at closure, the caecum would makecontact with the abdominal wall. Several areas of the caecum are gentlyabraded using a sterile, scalpel blade so that a homogenous surface ofpetechial hemorrhages are created. The reflected abdominal wall is alsoabraded, and the abraded areas exposed to air for 10 minutes.

Apposing portions of caecum and abdominal wall are either placed incontact with each other, or are apposed with each other with a measuredamount of an antiadhesion membrane placed between them. After coveringthe abraded areas, the surgical incisions were closed. 3 days to 4 weekslater, the animals are sacrificed using excess anesthetic, and the sitesof surgery exposed.

Adhesions are graded according to the method of Becker et al., J. Amer.Coll. Surgeons 183(4):297-306 (1996) from 0 to 3, with 0 being nodetectable adhesions, 1 having filmy thickness, avascular, grade 2having moderate thickness and limited vascularity, and grade 3 havingdense thickness and being well vascularized. Other methods of gradingadhesions may be used. (E.g., Diamond, Fertility and Sterility66(6):904-910 (1996); Interceed (TC7) Adhesion Study Group, Fertilityand Sterility 51(6):933(1989).

The bioresorbability of the membranes is determined at the time ofsacrifice by palpating the surgical sites and determining the presenceor absence of intact membrane. If intact membrane is present, it will beremoved from the site, and wet and dry weights will be determined. Themembranes of the invention are tailored to exhibit desired antiadhesionproperties.

Types of Surgery

Any type of surgical procedure benefits from the use of the membranes ofthe present invention. The following are exemplary, and are not intendedto be limiting.

Example 17 Cranial Surgery

For craniotomy use, membranes of the present invention are used as adural replacement graft following skull trephination and dural excision.The membrane is placed on the exposed cortex. The replacement of bone,closure of soft tissues and scalp completes the operation. The membraneforms a barrier to adhesion formation between the cortex and the skulland a scaffold to effect early ingrowth necessary for dural repair.

Example 18 Ocular Surgery

Ocular uses include surgery for glaucoma filtering. Successful glaucomafiltering surgery is characterized by the passage of aqueous humor fromthe anterior chamber through a surgically created fistula to thesubconjunctival space, which results in the formation of a filteringbleb. Bleb failure most often results from fibroblast proliferation andsubconjunctival fibrosis. To prevent this fibrosis, a membrane of thepresent invention can be placed post-operatively in the subconjunctivain the bleb space and a membrane also placed in the fistula.

Example 19 Musculoskeletal Surgery

Repair of tendon flexors can be enhanced by using membranes of thepresent invention, In tendon repair, collagen secreted by fibroblastsunites the ends of tendons. Adhesion formation usually binds the tendonto other tissue structures, obliterating the normal space between thetendon and tendon sheath, thereby interfering with the gliding functionnecessary for smooth movement. To prevent adhesions from forming betweenthe tendon and the sheath, a membrane of the present invention iswrapped around the reattached sutured tendon ends and/or a hydrogel formof the present invention is injected within the sheath.

For lumbar laminectomy and discectomy, a midline incision is made intothe lumbodorsal fascia just lateral to the bulbous tips of the spinousprocess. The paraspinous facia is opened to expose the interlaminar areaof the affected intervertebral disc. A laminectomy is performed toexpose the ligamentum flavum which is opened, exposing the dura. Thedura is retracted medially and the nerve root is identified andretracted. The disc area is exposed and explored with a nerve hook. Thetexture of the annulus, amount of bulge, presence of hernias or presenceof a hole in the annulus is determined. Disc removal is usuallyperformed through a small hole in the annulus. Post-surgical adhesionsare prevented by injecting a hydrogel form of the present invention intothe space around the annulus, nerve root, dura and laminectomy defect atthe conclusion of the procedure, before closing.

Example 20 Abdominal Surgery

Post-surgical adhesions are reported to form in up to 93% of previouslyoperated laparotomy patients. A laparotomy is required to gain access tothe abdomen for large and small intestine procedures, stomach,esophageal, and duodenal procedures, cholecystectomy and operations onthe female reproductive systems. In 1992, the Center for HealthStatistics reported 344,000 operations in the United States for lysis ofperitoneal adhesions. Peritoneal adhesions become pathologic when theyanatomically distort abdominal viscera producing various morbiditiesranging from intestinal obstruction and volvulus to infertility.Unfortunately, adhesion reformation and recurrence of intestinalobstruction following surgical division of adhesions is fairly common.

To prevent do novo adhesion formation or adhesion reformation, membranesof the present invention are placed directly over or wrapped around thesurgical site separating this site from the omentum. When closing,membranes of the present invention are placed under the midline incisionbetween the fascia and peritoneum. In laparoscopic procedures, ahydrogel form of the present invention is used to coat the surgical siteand trocar entry areas.

Example 21 Gynecological Surgery: Myomectomy via Laparotomy orLaparoscopy

The uterus is exposed and incised to remove the fibroid. The uterus isclosed with absorbable sutures. Posterior uterine incisions areassociated with more and a higher degree of adnexal adhesions than thatwith fundal or anterior uterine incisions. For posterior incisions,apply membranes of the present invention over the posterior uterineincision and beneath the anterior abdominal wall incision in order toprevent adhesion formation between the uterus and surrounding tissues.Anterior incisions more commonly result in adhesion formation betweenthe bladder an anterior wall of the uterus. Membranes of the presentinvention are placed over the anterior incision and between the uterusand bladder.

Example 22 Thoracic Surgery: Cardiac Procedures

Reoperative cardiac surgical procedures are becoming more commonplaceand result in the need to reduce or prevent postoperative mediastinaland pericardial adhesions. A median sternotomy precedes a midlinepericardiatomy. The pericardium is suspended, so that the heart andpericardial space are widely exposed. Dissection is performed. To createthe bypass, distal anastomoses are constructed using internal mammaryarteries, radial arteries, gastroepiploic arteries or saphenous veingrafts. In order to prevent adhesion formation, membranes of the presentinvention are wrapped around the anastomoses and placed between thepericardium and sternum before closing.

Other features, aspects and objects of the invention can be obtainedfrom a review of the figures and the claims. All citations herein areincorporated by reference in their entirety.

It is to be understood that other embodiments of the invention can bedeveloped and fall within the spirit and scope of the invention andclaims.

We claim:
 1. A method for preparing an association complex of CPS and PEcomprising the steps of:a) forming a solution of CPS and PE having a pHin the range of about 3.5 to about 6.0; b) allowing the solution to drythereby forming a dried from of the association complex; and c)increasing the pH of the dried complex.
 2. The method of claim 1,wherein in the step of increasing the pH increases the pH to about 6.0.3. The method of claim 1, wherein the step of increasing the pH includesthe use of a phosphate buffer to raise the pH to at least 6.0.
 4. Themethod of claim 1, wherein the solution of CPS and PE is a castingsolution and forming step includes forming a membrane.
 5. A method forpreparing an association complex for implantation at a surgical site inorder to prevent adhesion, comprising the steps of:a) forming a solutioncomprising CPS and a PE; b) acidifying said solution to a pH in therange of about 3.5 to about 6.0 in order to increase the residence timeat the surgical site; c) allowing the acidic association complex to atleast partially dry; and d) increasing the pH of the at last partiallydried association complex in order to increase biocompatability.
 6. Acomposition comprising an acidified association complex of CPS and PEwhich has been at least partially dried and made less acidic.
 7. Themethod of claim 5, wherein the CPS is CMC and the PE is PEO.
 8. Theassociation complex of claim 6, wherein the CPS is CMC and the PE isPEO.
 9. The association complex of claim 1 dried into a flexiblemembrane.
 10. An association complex of CPS and PE having a pH in therange of about 3.5 to about 6.0.
 11. The complex of claim 10, whereinthe degree of substitution of the CPS is above 0.5.
 12. The complex ofclaim 10, wherein the degree of carboxyl substitution of the CPS hasbeen increased in order to increase bioadhesiveness.
 13. The complex ofclaim 10, which is bioresorbable.
 14. The complex of claim 13 whichpersists in an animal for at least 48 hours but less than 29 days. 15.The complex of claim 10, wherein said complex is bioadhesive and whereinthe degree of substitution of the CPS is in the range of greater thanabout 0 to about 3.0 and/or the amount of PE is in the range of about90% to about 5%.
 16. A method of reducing post-surgical adhesions,comprising the steps ofa) accessing a delivery site; and b) deliveringto the delivery site an association complex made of a CPS and a PEhaving a pH in the range of about 3.5 to about 6.0, wherein theassociation complex has at least one property selected from the groupconsisting of bioresorbability, bioadhesiveness, antithrombogenicity,and antiadhesion.
 17. The method of claim 16, wherein the delivery siteis selected from the group consisting of orthopedic, gastrointestinal,abdominal, thoracic, cranial, cardiovascular, gynecological,arthroscopic, urological, dermal, subdermal, and musculoskeletal.
 18. Amethod of manufacturing an association complex of CPS and PE, consistingessentially of the following steps:a) selecting an aqueous solution of aCPS and an aqueous solution of a PE; b) mixing said solution of the CPSwith said solution of the PE, forming a mixed solution of the CPS andthe PE; and c) adjusting the pH of the mixed solution of the CPS and thePE to within the range of about 3.5 to about 6.0.
 19. An associationcomplex made of a CPS and a PE comprising from about 10% to about 95%CPS and from about 5% to about 90% PE, and made from a solution having apH in the range of about 3.5 to about 6.0.
 20. The association complexof claim 10 further comprising a drug.
 21. The association complex ofclaim 20, wherein the drug is selected from the group consisting ofantibiotics, anti-inflammatory drugs, hormones, chemotactic factors,analgesics, and anesthetics.
 22. An association complex of a CPS and aPE that possesses one or more of the properties selected from the groupconsisting of (1) a molecular weight of the CPS in the range of about100 kd to about 10,000 kd, (2) a molecular weight of the PE in the rangeof about 5 kd to about 8,000 kd, (3) a degree of substitution of the CPSin the range of greater than about 0 to about 3.0, (4) a percentage ofthe CPS in the range of about 10% to about 95%, (5) a percentage of PEin the range of about 5% to about 90%, and (6) a pH in the range ofabout 3.5 to about 6.0.
 23. The method of claim 1, wherein the step ofincreasing the pH includes use of a solution less acidic than saidsolution of CPS and PE to raise the pH.
 24. An association complex madeof a CPS and a PE having a pH in the range of about 3.5 to about 6.0,wherein the association complex has at least one property selected fromthe group consisting of bioresorbability, bioadhesiveness,antithrombogenicity, and antiadhesion.